US7674611B2 - Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase - Google Patents
Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase Download PDFInfo
- Publication number
- US7674611B2 US7674611B2 US11/569,339 US56933907A US7674611B2 US 7674611 B2 US7674611 B2 US 7674611B2 US 56933907 A US56933907 A US 56933907A US 7674611 B2 US7674611 B2 US 7674611B2
- Authority
- US
- United States
- Prior art keywords
- ethylenediamine
- disuccinate
- residue
- substitution
- seq
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
Definitions
- the present invention relates to a novel protein having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity and a gene encoding such protein. Further, the present invention relates to a modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase that can be derived as a mutant of the enzyme; a gene encoding the modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase; a recombinant DNA containing a gene DNA encoding the modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase; a transformant or transductant bearing the recombinant DNA containing the gene encoding the modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase; and a method for producing diaminoalkylene-N,N′-disuccinates by using such
- Diaminoalkylene-N,N′-disuccinates are important as intermediates of synthesis of medicines and agricultural chemicals, and have a unique property to capture heavy metals. Therefore, optically active forms of such compounds, which may be susceptible to biodegradation once released in nature, are expected to be potentially useful as chelating agents, builders for detergents, etc.
- Fumarase is an enzyme used to produce malic acid by adding water to fumaric acid. Therefore, when diaminoalkylene-N,N′-disuccinates are prepared by using microorganisms having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity, a fumarase in the microbial cells needs to be inactivated for preventing generation of by-products such as malic acid and the like.
- the inactivation rate of fumarase depends on the treatment temperature.
- the higher is the treatment temperature the faster the inactivation.
- the stability of fumarase varies with host microorganisms, but even when a microorganism whose fumarase is not inactivated readily is used, a higher treatment temperature is preferred. Accordingly, the objective of the present invention is to provide an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase having improved heat resistance.
- the present invention includes:
- a protein comprising an amino acid sequence described in SEQ ID NO: 1, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by deletion, substitution, or addition of one or more amino acid residues, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least one amino acid residue of a lysine residue at 120, an isoleucine residue at 166, and an alanine residue at 365 with a different amino acid residue, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least the isoleucine residue at 166 with serine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least an isoleucine residue at 166 with threonine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid and an isoleucine residue at 166 with serine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid and an isoleucine residue at 166 with threonine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least an isoleucine residue at 166 with serine and an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least an isoleucine residue at 166 with threonine and an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid and an isoleucine residue at 166 with serine an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- a protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid and an isoleucine residue at 166 with threonine and an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
- the gene according to (16) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by deletion, substitution or addition of one or more bases.
- a gene encoding the protein according to (3) (19) The gene according to (18) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of at least one base of bases from 358 to 360, from 496 to 498, and from 1093 to 1095 with a different base. (20) A gene encoding the protein according to (4). (21) The gene according to (20) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG.
- the gene according to (23) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of the base, thymine, at 497 with guanine.
- a gene encoding the protein according to (6) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases ATC from 496 to 498 with ACN (N refers to A, G, C or T).
- N refers to A, G, C or T.
- the gene according to (26) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, thymine, at 497 with cytosine.
- a gene encoding the protein according to (7) A gene encoding the protein according to (7).
- the gene according to (29) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T).
- the gene according to (29) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, cytosine, at 1094 with thymine.
- (32) A gene encoding the protein according to (8).
- the gene according to (32) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG, and bases ATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T).
- the gene according to (32) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, adenine, at 358 with guanine, and a base, thymine, at 497 with guanine.
- the gene according to (35) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG, and bases ATC from 496 to 498 with ACN (N refers to A, G, C or T), respectively.
- the gene according to (35) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, adenine, at 358 with guanine, and a base, thymine, at 497 with cytosine, respectively.
- the gene according to (38) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases ATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T), and bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T), respectively.
- the gene according to (38) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, thymine, at 497 with guanine, and a base, cytosine, at 1094 with thymine, respectively.
- (41) A gene encoding the protein according to (11).
- the gene according to (41) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases ATC from 496 to 498 with ACN (N refers to A, G, C or T), and bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T), respectively.
- the gene according to (41) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, thymine, at 497 with cytosine, and a base, cytosine, at 1094 with thymine, respectively.
- the gene according to (44) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG, bases ATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T), and bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T), respectively.
- the gene according to (44) comprising a nucleotide sequence wherein the base, adenine, at 358 is substituted with guanine, the base, thymine, at 497 with guanine, and the base, cytosine, at 1094 with thymine in the nucleotide sequence of SEQ ID NO: 2.
- (47) A gene encoding the protein according to (13).
- the gene according to (47) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, adenine, at 358 with guanine, a base, thymine, at 497 with cytosine, and a base, cytosine, at 1094 with thymine, respectively.
- a recombinant wherein the gene DNA according to any one of (14) to (49) is inserted into a DNA vector.
- a transformant or transductant comprising the recombinant DNA according to (50).
- a method of producing an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase comprising a step of culturing the transformant or transductant according to (51) to collect the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase from the resulting culture.
- a method of producing a diaminoalkylene-N,N′-disuccinate comprising a step of reacting fumaric acid and a diamine in the presence of the transformant or transductant according to (51) to collect the diaminoalkylene-N,N′-disuccinate from the resulting reaction products.
- FIG. 1 is a restriction map of the plasmid pEDS9001.
- FIG. 2 is a restriction map of the plasmid pEDS9003.
- FIG. 3 is a restriction map of the plasmid pEDS9020.
- FIG. 4 is an assembly scheme of the expression vector pFY529V.
- FIG. 5 is a graph for evaluating heat resistance of the single mutants.
- FIG. 6 is a schematic representation to illustrate the constructions of the single mutants and the multiple mutants: square indicates the mutations of T497C (Ile166Thr), triangle indicates the mutations of A358G (Lys120Glu), circle indicate the mutations of T497G (Ile166Ser), and diamond indicates the mutations of C1094T (Ala365Val), respectively.
- FIG. 7 is a graph for evaluating heat resistance of the multiple mutants.
- the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase refers to an enzyme capable of reversibly producing ethylenediamine-N,N′-disuccinate from fumaric acid and ethylenediamine, but it may produce ethylenediamine-N-monosuccinic acid depending on reaction conditions. Further, the present enzyme also reacts on other diamines in addition to ethylenediamine, producing corresponding diaminoalkylene-N,N′-disuccinates.
- wild type means that an amino acid sequence that constitutes an enzyme kept in a microorganism separated from nature, and a nucleotide sequence encoding such an enzyme are, either intentionally or unintentionally, not lost, deleted, inserted, or substituted with other amino acids or bases.
- the present inventors conducted screening of microorganisms having such an enzyme activity for further useful ethylenediamine-N,N′-disuccinate:ethylenediamine lyase.
- MR-E001 Brevundimonas diminuta strain MR-E001 (hereinafter also referred to the MR-E001 strain), which has a high activity of the enzyme, was isolated, and an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene was obtained from the MR-E001 strain.
- MR-E001 Brevundimonas diminuta strain MR-E001 (hereinafter also referred to the MR-E001 strain)
- an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene was obtained from the MR-E001 strain.
- it was discovered that in the amino acid sequence of the enzyme by substituting at least one or more amino acid residues with residues selected from the group of natural amino acids,
- the modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase of the present invention can be obtained, for example, by the following method.
- a wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene is obtained.
- any known technique can be used.
- chromosomal DNA prepared from the MR-E001 strain is used as a template and PCR (Polymerase Chain Reaction) is conducted to obtain a DNA fragment containing a part of the gene.
- degenerate primers can be used, which are designed based on the amino acid sequence obtained by amino acid analysis after isolation and purification of the enzyme. Further, when the sequence of the gene of interest is expected to be homologous with a sequence of the gene derived from a different species that has been already known, or when a gene homologous with a sequence of the gene derived from a different species that has been already known is to be obtained, degenerate primers can be designed for PCR according to the amino acid sequence information encoded by such a known gene derived from the different species. By using the primers designed thereby, the chromosomal DNA from the MR-E001 strain is used as a template and PCR is conducted, and the resulting amplified DNA product is used as a probe for colony hybridization that will be conducted later.
- a DNA library is prepared.
- the chromosome from the MR-E001 strain prepared according to a known method, for example, the method made by Saito and Miura [Biochem. Biophys. Acta, 72, 619 (1963)], is cleaved or partially cleaved by a suitable restriction enzyme, which is then ligated to a vector DNA that has been treated by a restriction enzyme that can generate cleaved terminals linkable to such cleaved terminals of the restriction enzyme described above, so that transformants or transductants of a suitable microorganism host are formed, and thereby the DNA library of the chromosome is produced.
- Examples of the microorganisms that can be hosts for the transformants or the transductants include, when Escherichia coli is used, E. coli strain K12, strain JM109, strain XL1-Blue, etc., but should not be particularly limited to these.
- Examples of the plasmid DNA used for forming the transformants include, when E. coli is the host, pBR322, pUC18, pBluescript II SK(+), etc., which have auto-replicable regions in E. coli .
- the vector DNA should not be limited to the above plasmid vector DNAs, but phage vector DNAs may be used to form transductants.
- transformants when plasmid vector DNAs are used, and transductants when phage vector DNAs are used. All of the above transformants and transductants are included in the present invention. Examples, wherein transformants are created utilizing plasmid vector DNAs, are described below.
- Colony hybridization is conducted for the resulting DNA library of the chromosome, using the PCR-amplified DNA product described above as a probe.
- the colony hybridization may be an ordinary method.
- the one described below can be used.
- transformants from the chromosomal DNA library grown on an agar medium are transferred to a nylon membrane, and then the DNA is fixed by cytolysis.
- the amplified DNA product by PCR described above is rendered to be a probe by labeling, for example, using a DNA Labeling kit (from Roche Diagnostics), and then hybridized with the membrane.
- Positive clones can be selected, for example, by using a DNA Luminescent Detection kit (from Roche Diagnostics).
- plasmid DNA is prepared according to the ordinary method, optionally subcloning is conducted, and then nucleotide sequences of the inserted fragments are determined. Any method can be used for the determination of the nucleotide sequences, and usually the nucleotide sequences can be determined by the dideoxy method (Methods in Enzymology, 101, 20-78, 1983) using a commercial kit or the like.
- the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene derived from the MR-E001 strain can be obtained, and also an amino acid sequence and a nucleotide sequence thereof can be determined.
- a modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase wherein in the amino acid sequence of the resulting wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase, at least one amino acid residue is substituted with a different natural amino acid residue, any method can be used and usually a well known method can be used.
- examples of methods for treating the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene DNA include: to contact and react a mutagenic chemical such as hydroxylamine and nitrous acid; to induce mutations by irradiating ultraviolet rays; to induce mutations on a random basis by using PCR; to generate site-specific substitutions by utilizing a commercial kit; to selectively cleave gene DNA and then remove and add selected oligonucleotides for ligation; and the like.
- transformants is formed.
- the plasmid vectors that can be used for transformation for example, when E.
- coli is the host, those plasmid vectors included in the step for preparing the DNA library described above may be used, but it is preferable to use an expression vector with high expression efficiency, such as an expression vector, pKK233-2 (from Amersham), which has a trc promoter, or a derivative of pKK233-2, i.e., pFY529V, which will be described later in an example, in order to efficiently detect a remaining activity of ethylenediamine-N,N′-disuccinate:ethylenediamine lyase after heat treatment in the later step for screening.
- an expression vector with high expression efficiency such as an expression vector, pKK233-2 (from Amersham), which has a trc promoter, or a derivative of pKK233-2, i.e., pFY529V, which will be described later in an example, in order to efficiently detect a remaining activity of ethylenediamine-N,N′-disuccinate:ethylenediamine ly
- the vectors and the hosts used in the present invention should not be limited to those plasmids described above and E. coli .
- Examples of the vectors include plasmid DNA, bacteriophage DNA, retrotransposon DNA, artificial chromosomal DNA, etc.
- the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene DNA of the present invention requires genes to be incorporated into a vector, so that the gene DNA can be expressed in a host thereof. Therefore, to the vector of the present invention, in addition to the gene DNA of the present invention, a promoter, a terminator, an enhancer, a splicing signal, a poly-A addition signal, a selection marker, a ribosome-binding sequence (SD sequence), etc., can be linked. Further, examples of the selection markers include a dihydrofolate reductase gene, an ampicillin resistance gene, a neomycin resistance gene, etc.
- the transformant of the present invention can be obtained by transferring the recombinant vector of the present invention into a host in a manner that the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene can be expressed.
- the hosts include bacteria such as E. coli, Bacillus subtilis , etc.
- yeast, animal cells, insect cells, plant cells, etc. can be also used.
- bacteria from the genus of Escherichia examples include Escherichia coli , etc.
- bacteria from the genus of Bacillus examples include Bacillus subtilis , etc.
- Any technique for transferring DNA into bacteria can be used. Examples of such techniques include: to utilize calcium ions; electroporation; and the like.
- yeast When yeast is the host, for example, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris , etc., are used. There should be no particular restriction on how recombinant vectors are transferred into yeast, and any technique for transferring DNA into yeast can be used. Examples of such techniques include: electroporation; the spheroplast method; the lithium acetate method; and the like.
- monkey COS-7 cells When animal cells are the host, monkey COS-7 cells, Vero cells, CHO cells; mouse L cells; rat GH3 cells; human FL cells; and the like are used.
- Examples of techniques for transferring recombinant vectors into animal cells include: electroporation; the Ca-phosphate method; lipofection; and the like.
- insect cells When insect cells are the host, Sf9 cells, Sf21 cells, and the like are used.
- techniques for transferring recombinant vectors into insect cells include: the Ca-phosphate method; lipofection; electroporation; and the like.
- plant cells When plant cells are the host, examples include corn, rice, tobacco, and the like, but should not be limited to these.
- Examples of technique for transferring recombinant vectors into plant cells include: the Agrobacterium method; the particle gun method; the PEG method; electroporation; and the like.
- the resulting transformants are cultured on an agar medium to form colonies followed by liquid culture to produce an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase.
- the resulting culture for example, is subjected to heat treatment for 30 min. at 40 to 65° C. followed by the determination of remaining ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activities.
- a transformant with a higher remaining ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity is selected.
- a nucleotide sequence of the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene inserted into the recombinant DNA of the resulting superior transformant thereby can be determined, for example, by the dideoxy method.
- the nucleotide sequence of the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene DNA of the present invention is represented by SEQ ID NO: 2, and an amino acid sequence encoded by the gene of the present invention is represented by SEQ ID NO: 1.
- a mutated enzyme whose heat resistance is more improved than a single mutant by combining plural different single-substitution mutations, each of which provides heat resistance to the enzyme, to form a multiple mutant.
- Any technique can be used for forming a multiple mutant, and examples include: to generate a site-specific substitution by using an artificial single-strand oligonucleotide; to cleave a DNA fragment containing multiple different single mutated sites by restriction enzymes, which are then ligated; and the like.
- amino acids in the amino acid sequence of SEQ ID NO: 1 may be deleted from the amino acid sequence of SEQ ID NO: 1; from 1 to 10, preferably from 1 to 5, amino acids may be added to the amino acid sequence of SEQ ID NO: 1; or from 1 to 10, preferably from 1 to 5, amino acids in the amino acid sequence of SEQ ID NO: 1 may be substituted with other amino acids.
- amino acid of Lys at 120, Ile at 166, and Ala at 365 is substituted by a different amino acid in the amino acid sequence of SEQ ID NO: 1.
- the amino acid substitutions at the above three locations can be optionally combined. Aspects of preferred substitutions are shown below, wherein in the following aspects of substitutions, numerals refer to location numbers in the amino acid sequence of SEQ ID NO: 1; letters in the left side of the numbers refer to amino acids (in single letters) before substitution; and letters in the right side of the numbers refer to amino acids (in single letters) after substitution.
- the locations in the nucleotide sequence of SEQ ID NO: 2 corresponding to Lys at 120 are from 358 to 360, and the nucleotide sequence is “AAA.”
- the codon for glutamic acid is GAA or GAG.
- bases can be substituted so that AAA becomes GAA or GAG.
- a at 358 is substituted with G (AAA ⁇ GAA).
- the bases ATC from 496 to 498 can be substituted with AGC, AGT, ACA, ACC, ACG, or ACT.
- T at 497 is substituted with G (ATC ⁇ AGC).
- the bases ATC from 496 to 498 can be substituted with ACA, ACC, ACG, or ACT.
- T at 497 is substituted with C (ATC ⁇ ACC).
- the bases GCC from 1093 to 1095 can be substituted with GTA, GTG, GTC, or GTT.
- C at 1094 is substituted with T (GCC ⁇ GTC).
- the amino acids after substitution should not be limited to the above examples.
- at least one amino acid residue at 120, 166, or 365 in the amino acid sequence of SEQ ID NO: 1 is substituted with a different amino acid from the examples above, at least one base of the codons encoding such amino acids can be substituted with another base.
- ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity refers to a catalytic activity to produce a diaminoalkylene-N,N′-disuccinate by reacting fumaric acid and a diamine.
- a gene that hybridizes under stringent conditions to DNA made of a sequence complementary to the DNA made of the nucleotide sequence of SEQ ID NO: 2 or a part of such a sequence, and also encodes a protein having the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity is included in the gene of the present invention.
- the stringent conditions refer to typical conditions in which specific hybridization occurs.
- Examples include conditions where highly homologous nucleic acids, i.e., DNAs with 80% or more, preferably 90% or more, homology, also each DNA encoding a protein having the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity, hybridize each other, whereas DNAs with less homology do no hybridize each other. More specifically, it refers to conditions in which the sodium concentration is from 300 to 2000 mM, preferably from 600 to 900 mM, and the temperature is from 40 to 75° C., preferably from 55 to 65° C.
- the gene DNA of the present invention can be thereafter obtained by PCR, wherein chemically synthesized or cloned DNA is a template, or by hybridization using a DNA fragment having such a nucleotide sequence as a probe.
- the heat resistance refers to a property capable of retaining the enzyme activity even in the temperature range where the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase is deactivated.
- a temperature range is from 45 to 60° C., preferably from 50 to 60° C.
- modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase can retain 60% of the enzyme activity exhibited at 50° C.
- the resulting transformant bearing the recombinant plasmid DNA containing the modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene wherein a single mutation or multiple mutations are transferred can be used to produce an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase.
- the above transformant e.g., an E. coli transformant
- the above transformant can be cultured to collect the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase from such a culture.
- the term “culture” refers to any of culture supernatant, cultured cells or cultured cell mass; or destructed cells or cell mass.
- an ordinary method used for culturing a host is followed.
- any medium, natural or synthetic, containing a carbon source, a nitrogen source, inorganic salts, etc., that can be utilized by the microorganism and capable of efficiently culturing the transformant can be used.
- carbon sources include carbohydrates such as glucose, fructose, sucrose, starch; organic acids such as acetic acid, propionic acid; and alcohols such as ethanol, propanol.
- nitrogen sources include ammonia; ammonium salts of inorganic and organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, and other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor, and the like.
- inorganic salts include potassium dihydrogenphosphate, potassium hydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate.
- the culture is usually performed under aerobic conditions such as shake culture, or culture by aeration with stirring. With a solution containing an inorganic or organic acid, or an alkali, the pH is adjusted.
- an antibiotic such as ampicillin or tetracycline may be optionally added to the medium.
- an inducer may be optionally added to the medium.
- an inducer may be optionally added to the medium.
- a microorganism transformed with an expression vector having a promoter that is inducible with isopropyl- ⁇ -D-thiogalactoside (IPTG) is cultured, IPTG, etc.
- IPTG isopropyl- ⁇ -D-thiogalactoside
- IAA indoleacetic acid
- Examples of media for culturing a transformant obtained from an animal cell host include media such as general ones, e.g., a RPMI1640 medium and a DMEM medium; or a medium wherein fetal bovine serum, etc., are added to a RPMI1640 or DMEM medium.
- the culture is usually performed under 5% CO 2 at 37° C. for from 1 to 30 days.
- an antibiotic such as kanamycin or penicillin may be optionally added to the medium.
- the protein of the present invention When the protein of the present invention is produced within the cell mass or cells following the culture, the protein of interest is collected by destructing the cell mass or the cells by ultrasonic, repeated freeze and thawing, homogenizing, etc. Further, when the protein of the present invention is produced outside a microorganism or cells, the culture solution per se is used, or the microorganism or the cells are removed by centrifugation or the like. Then, the protein of the present invention can be isolated and purified by an ordinary biochemical technique used for protein isolation and purification. Examples of such techniques include ammonium sulfate precipitation, gel chromatography, ion-exchange chromatography, and affinity chromatography, which may be used alone or in any proper combination thereof.
- the culture can be performed by using an ordinary plant culture medium, for example, an MS basal medium, LS basal medium, etc. Any ordinary culture method, either liquid culture or solid culture, can be used.
- cells are first destructed by cytolysis using an enzyme such as cellulase and pectinase, ultrasonic destruction, grinding, and the like. Then, insoluble matters are removed by filtration or centrifugation to obtain a crude protein solution.
- the protein of the present invention is purified from the above crude solution by salt precipitation, chromatography of various types (e.g., gel filtration chromatography, ion-exchange chromatography, affinity chromatography, etc.), SDS-polyacrylamide gel electrophoresis, and the like, or optionally the combination thereof.
- a medium for the culture for example, containing one or more nitrogen sources such as yeast extract, tryptone, polypeptone, corn steep liquor, percolate of soy bean and wheat bran loaded with one or more inorganic salts such as sodium chloride, potassium dihydrogenphosphate, potassium hydrogenphosphate, magnesium sulfate, magnesium chloride, ferric chloride, ferric sulfate, and manganese sulfate, and optionally further with sugar materials, vitamins, etc., is used.
- the initial pH of the medium is suitably adjusted from 7 to 9.
- submerged culture, shake culture, stationery culture, or the like is performed from 25 to 42° C. for from 6 to 24 hrs.
- the resulting microbial cells are harvested, washed with a suitable buffer solution, for example, a 50 mM boric acid buffer solution (pH 9.0), and then suspended in the buffer solution to prepare a cell suspension.
- a suitable buffer solution for example, a 50 mM boric acid buffer solution (pH 9.0)
- a fumarase activity is lost, and microbial mass having the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity can be prepared.
- aqueous solutions of optically active S,S-diaminoalkylene-N,N′-disuccinates that do not contain by-products such as malic acid can be prepared.
- Brevundimonas diminuta strain MR-E001 was deposited on Feb. 5, 2003, with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), under the Accession No. FERM BP-08677.
- the MR-E001 strain was cultured with shaking in 100 ml of an EDDS medium [0.2% ethylenediamine-N,N′-disuccinate, 0.2% glucose, 0.1% Bacto yeast extract, 0.05% polypeptone, 0.1% MgSO 4 .7H 2 O, 25% (v/v) phosphate buffer (1M, pH 7.0), a 0.5% (v/v) mixture solution of metal salts (containing 56 g of NaSO 4 , 8 g of MgCl 2 .6H 2 O, 0.8 g of CaCl 2 , 0.6 g of MnSO 4 .4H 2 O, 0.12 g of FeCl 3 .6H 2 O, and 0.06 g of ZnSO 4 per 100 ml)] at 30° C.
- an EDDS medium 0.0.2% ethylenediamine-N,N′-disuccinate, 0.2% glucose, 0.1% Bacto yeast extract, 0.05% polypeptone, 0.1% MgSO 4 .7H 2
- saline-EDTA solution 0.1 M EDTA, 15 M NaCl, pH 8.0
- the resulting suspension was shaken at 37° C. for 1 hr. and then frozen.
- 10 ml of a Tris-SDS solution 1% SDS, 0.1 M NaCl, 0.1 M Tris, pH 9.0
- proteinase K from Merck
- TE-saturated phenol (TE:10 mM Tris, 1 mM EDTA, pH 8.0) was added thereto and stirred followed by centrifugation. The upper layer was collected, to which a two-fold volume of ethanol was added, and then DNA was collected by rolling with a glass rod followed by removal of phenol using 90%, 80%, and 70% ethanol in this order. Subsequently, the DNA was dissolved in 3 ml of a TE buffer, to which a ribonuclease A solution (that has been treated by heat at 100° C. for 15 min.) was added at the final concentration of 10 mg/ml to shake at 37° C. for 30 min.
- a ribonuclease A solution that has been treated by heat at 100° C. for 15 min.
- Proteinase K was further added to shake at 37° C. for 30 min. Then, an equal volume of TE-saturated phenol was added thereto, which was separated by centrifugation into upper and lower layers to collect the upper layer (hereinafter, this procedure is referred to as phenol extraction). After phenol extraction was repeated twice, an equal volume of chloroform (containing 4% isoamyl alcohol) was added to repeat the similar extraction procedure twice (hereinafter, this procedure is referred to as chloroform extraction). Next, ethanol twice in volume thereof was added to the upper layer, and the DNA was recovered by rolling with a glass rod to obtain a chromosomal DNA sample.
- chloroform containing 4% isoamyl alcohol
- the present applicants previously succeeded in isolating an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene from Brevundimonas sp. strain TN-3, and determined for the first time the amino acid sequence and the gene sequence thereof [JP Patent Publication (Kokai) No. 10-210984A (1998)].
- the degenerate primers used in the above publication i.e., artificial DNAs having the sequences represented by SEQ ID NO: 3 and SEQ ID NO: 4 (Primer #1 and Primer #2, respectively) were used, and PCR was conducted by using the chromosomal DNA from the MR-E001 strain obtained in step (1).
- Primer #1 ATGACICCIC AYAAYCCIGA YGC (SEQ ID NO: 4) Primer #2: CCDATYTGCAT YTTICCIGC RACIGAICCD ATYTC
- 1 ⁇ l of the chromosomal DNA from the MR-E001 strain, 10 ⁇ l of 10 ⁇ buffer for the reaction, 4 ⁇ l of 10 mM dNTP, 1 ⁇ l of Primer #1 and 1 ⁇ l of Primer #2 (equivalent to 100 pmol, respectively), and 1 ⁇ l of ExTaq (from Takara Shuzo) were admixed to obtain a 100 ⁇ l solution.
- the resulting solution was incubated at 95° C. for 30 sec. (denaturation), at 55° C. for 30 sec. (annealing), and at 72° C. for 2 min. (extension) for 30 cycles.
- phenol extraction and chloroform extraction were performed to recover the amplified DNA by ethanol precipitation.
- the resulting DNA was separated by 1.0% agarose gel electrophoresis, and then obtained was a DNA fragment having about 300 bp, which was expected to encode a part of the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene of the MR-E001 strain.
- This DNA fragment obtained thereby was labeled by using a DIG DNA Labeling kit (from Roche Diagnostics) to provide a probe.
- the pUC18 fragment used for the ligation was prepared by the process described below. To 2 ⁇ l of a solution preserving pUC18, 5 ⁇ l of 10 ⁇ buffer for restriction enzyme, 40 ⁇ l of sterile water, and 3 ⁇ l of a restriction enzyme KpnI were added to react at 37° C. for 2 hrs., and then phenol extraction and chloroform extraction were performed followed by ethanol precipitation. The precipitate was dried, which then was dissolved into 50 ⁇ l of sterile water.
- alkaline phosphatase from Takara Shuzo
- 10 ⁇ l of 10 ⁇ buffer and 39 ⁇ l of sterile water to react at 65° C.
- phenol extraction and chloroform extraction were performed followed by ethanol precipitation.
- the precipitate was dried, which then was dissolved into sterile water.
- E. coli strain JM109 was inoculated into 1 ml of an LB medium (1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% NaCl) to pre-culture at 37° C. for 5 hrs. under aerobic conditions.
- an SOB medium 2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO 4 , 1 mM MgCl 2
- 0.4 ml of this culture was added to culture at 18° C. for 20 hrs.
- the resulting culture was divided into 200 ⁇ l aliquots, each of which was inoculated onto a LBamp medium (a LB medium containing 100 mg/l ampicillin and 1.5% agar) to culture at 37° C.
- a LBamp medium a LB medium containing 100 mg/l ampicillin and 1.5% agar
- transformants expected to carry the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene were selected by colony hybridization.
- the transformants grown on the agar medium were transferred on a nylon membrane (Biodyne A, from Nihon Pall), where microbial cells were lysed, and DNA was fixed.
- the fixed DNA was treated with the probe (about 300 bp) prepared in step (2) to select colonies having the recombinant DNA of interest by using a DIG Luminescent Detection Kit (from Roche Diagnostics).
- the transformant selected in step (4) was cultured in 100 ml of an LBAmp medium (an LB medium containing 100 mg/L ampicillin) at 37° C. overnight. After cells were harvested, the plasmid DNA was recovered by using a Flexi Prep (from Amersham Biosciences). The resulting recombinant plasmid DNA was named pEDS9001.
- the plasmid pEDS9001 obtained in step (5) was cleaved by several restriction enzymes to make a restriction map ( FIG. 1 ).
- the plasmid was subcloned by an ordinary method. Specifically, after pEDS9001 was cleaved by a restriction enzyme BamHI, agarose gel electrophoresis was performed to extract from the gel a DNA fragment of about 5.3 Kb, which was recovered by using a DNA PREP (from Diatron). After autoligation by using a DNA Ligation Kit Ver. 1 (from Takara Shuzo), E. coli strain JM109 was transformed to obtain a plasmid (pEDS9003) ( FIG. 2 ), wherein a fragment of about 2.6 Kb expected to contain the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene is inserted.
- a nucleotide sequence around the region defined in step (6) was determined by using a fluorescence sequencer ALFII (from Amersham Biosciences). As a result, the nucleotide sequence (SEQ ID NO: 2) encoding an open reading frame made of the amino acid sequence of SEQ ID NO: 1 was discovered.
- a fragment of about 2.6 Kb is prepared by agarose gel electrophoresis, which was inserted into an E. coli vector pUC 119.
- E. coli strain JM109 was transformed by using the prepared ligation solution to obtain a plasmid of interest.
- the thus prepared plasmid was named pEDS9020 ( FIG. 3 ), and the transformant was named JM109/pEDS9020.
- JM109/pEDS9020 and JM109/pEDS020 [described in JP Patent Publication (Kokai) No. 10-210984A (1998)] as a control was inoculated into 1 ml of an LBAmp medium to culture with shaking at 37° C.
- One enzyme unit (U) was defined as the amount of enzyme to produce 1 ⁇ mol of S,S-ethylenediamine-N,N′-disuccinate per minute under the above determination conditions.
- the activities of JM109/pEDS9020 and JM109/pEDS020 per cell (per OD630) were determined to be 1.22 mU/ODml and 0.89 mU/ODml, respectively. It was confirmed that the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase derived from the MR-E001 strain has a high activity of the enzyme.
- pEDS9020 was deposited on Feb. 5, 2003, with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), under the Accession No. FERM BP-08676.
- mutations were induced on a random basis into the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene.
- utilized was base substitution by nucleotide misincorporation by PCR.
- the oligonucleotide ED-01 (SEQ ID NO: 5), which contains the initiation codon region of the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene, and the oligonucleotide ED-02 (SEQ ID NO: 6), which contains the downstream region by about 50 bp from the termination codon of the gene, were primers for PCR inducing mutations, and 100 ⁇ l of a PCR reaction solution having a composition described below was prepared:
- reaction solution (SEQ ID NO: 6) ED-02: AAAC AAGCTT CGTCATGGCT ATCCCCTC (The underlined part is a cleavage recognition site by the restriction enzyme HindIII). Composition of reaction solution:
- Template DNA (pEDS9020 prepared in the above step) 1 ⁇ l 10 ⁇ PCR buffer (from GIBCO) 10 ⁇ l 50 mM MgCl 2 (from GIBCO) 3 ⁇ l Primer ED-01 1 ⁇ l Primer ED-02 1 ⁇ l 2.5 mM dNTP 2 ⁇ l each 10 mM dITP 2 ⁇ l 10 mM dBraUTP 2 ⁇ l Sterile water 71 ⁇ l Taq DNA polymerase (from GIBCO) 1 ⁇ l.
- the above reaction solution was incubated at 94° C. for 30 sec. (denaturation), and at 68° C. for 180 sec. (annealing and extension) for 30 cycles. After the above PCR was completed, an amplified fragment of about 1.5 kb was detected from 10 ⁇ l of the reaction solution by using 0.7% agarose gel electrophoresis.
- the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene was amplified by usual PCR to use it as a control for evaluating heat resistance. Specifically, 100 ⁇ l of a PCR reaction solution having a composition described below was prepared:
- Template DNA (pEDS9020) 1 ⁇ l 10 ⁇ Pyrobest Buffer (from Takara Shuzo) 10 ⁇ l Primer ED-01 1 ⁇ l Primer ED-02 1 ⁇ l 5 mM dNTP 2 ⁇ l each Sterile water 78 ⁇ l Pyrobest TM DNA polymerase (from Takara Shuzo) 1 ⁇ l.
- the above reaction solution was incubated at 94° C. for 30 sec. (denaturation), and at 68° C. for 180 sec. (annealing and extension) for 30 cycles. After the above PCR was completed, an amplified fragment of about 1.5 kb was detected from 10 ⁇ l of the reaction solution by using 0.7% agarose gel electrophoresis.
- the cleavage recognition site by the restriction enzyme NcoI, and the cleavage recognition site by the restriction enzyme HindIII were transferred, respectively (the underlined parts of the nucleotide sequences of Primer ED-01 and Primer ED-02).
- the amplified DNA product can be readily inserted, by the cleavage thereof using both of the restriction enzymes, into between the NcoI site and the HindIII of an expression vector pFY529V, which will be described later.
- an E. coli expression vector pFY529V which has a high copy number and great expression efficiency, was prepared ( FIG. 4 ).
- an expression vector pKK233-2 having a trc promoter from Amersham
- 1 ⁇ l of a restriction enzyme NaeI, 1 ⁇ l of a restriction enzyme ScaI, 1 ⁇ l of 10 ⁇ buffer for restriction digestion, and 2 ⁇ l of sterile water were added to perform cleavage reaction at 37° C. for 12 hrs.
- an NaeI-ScaI fragment (1.2 kb) that does not contain the replication origin of the plasmid was extracted by 0.7% agarose gel electrophoresis, and 3 ⁇ l of a TE solution (10 mM Tris, 1 mM EDTA, pH 8.0) containing the DNA fragment was recovered by using a DNA PREP (from Diatron).
- a DNA PREP from Diatron
- a PvuII-ScaI fragment (1.6 kb) that contains the replication origin of the plasmid was extracted by 0.7% agarose gel electrophoresis, and 1 ⁇ l of a TE solution containing the DNA fragment was recovered by using a DNA PREP (from Diatron). Both of the resulting DNA fragments were ligated by using a DNA Ligation Kit Ver. 1 (from Takara Shuzo).
- a reaction solution containing the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes wherein the mutations were induced that was obtained by PCR in step (1) was purified by ethanol precipitation according to the conventional method, and a precipitate was again suspended in 70 ⁇ l of sterile water. Thereto, 10 ⁇ l of 10 ⁇ buffer for restriction digestion, 10 ⁇ l of a restriction enzyme NcoI, and 10 ⁇ l of a restriction enzyme HindIII were added to perform cleavage reaction at 37° C. for 12 hrs. After the cleavage reaction, phenol extraction and chloroform extraction were conducted followed by ethanol precipitation.
- the precipitate was again suspended in 100 ⁇ l of sterile water to obtain a mutated DNA fragment solution.
- 67 ⁇ l of sterile water 10 ⁇ l of 10 ⁇ buffer for restriction digestion, 10 ⁇ l of a restriction enzyme NcoI, and 10 ⁇ l of a restriction enzyme HindIII were added to perform cleavage reaction at 37° C. for 12 hrs. After the cleavage, phenol extraction and chloroform extraction were conducted followed by ethanol precipitation for purification. Then, the precipitate was again suspended in 10 ⁇ l of sterile water to obtain a cleaved pFY529V solution.
- the mutated DNA fragments and the expression vector pFY529V were ligated by using a DNA Ligation Kit Ver. 1 (from Takara Shuzo). By admixing 3 ⁇ l of the above mutated DNA fragment solution, 1 ⁇ l of the above cleaved pFY529V solution, 16 ⁇ l of an A solution of the kit, and 4 ⁇ l of a B solution of the kit, the ligation was performed at 16° C. for 16 hrs. By using a reaction solution after the ligation, E. coli JM109 strain was transformed by the method described in Example 1 [step (4)] to obtain transformants bearing various mutation induced EDDSase genes.
- step (1) the similar procedure was performed wherein the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase gene that was amplified in step (1) was used as a fragment for insertion.
- a plasmid was extracted from the resulting transformant colony, and a nucleotide sequence thereof was confirmed by using a fluorescence sequencer ALF II (from Amersham Biosciences).
- pEDTrc9003 was deposited on Feb. 5, 2003, with International Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan), under the Accession No. FERM BP-08675.
- the JM109 transformants containing the mutated ethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes obtained in step (3), and JM109/pEDTrc9003 as a control were inoculated into an LBAmp medium, which had been dispensed in a 48-hole multi-dish in a 1.5 ml aliquot, for liquid culture at 37° C. for 12 hrs.
- the resulting culture was treated by heat at 50° C. for 30 min. followed by the determination of remaining ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity according to the method described in Example 2. About 10,000 strains of the transformants obtained in step (3) were screened.
- ethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes wherein the mutations were induced contained in the four candidate strains with improved heat resistance that were obtained in step (4) were analyzed in order to confirm what type of and where the mutation was induced according to the procedure described below.
- Recombinant plasmid DNAs contained in the four candidate strains with improved heat resistance were purified by using a Flexi Prep (from Amersham Biosciences), and the resulting recombinant plasmid DNAs were named pEDTrcI-2, pEDTrcI-23, pEDTrcJ-05, and pEDTrcK-01, respectively.
- mutated enzymes themselves contained in the respective plasmids were named I-2, I-23, J-05, and K-01.
- Nucleotide sequences of the mutated ethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes contained in these recombinant plasmid DNAs were determined by using a fluorescence sequencer ALF II (from Amersham Biosciences).
- coli bearing the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase JM109/pEDTrc9003 were inoculated into 1.5 ml of an LBAmp medium to incubate with shaking at 30° C. for 8 hrs. Then, 400 ⁇ l of each of the culture solutions was inoculated into 40 ml of an LBAmp medium prepared in a 500 ml Erlenmeyer's flask to culture with shaking at 37° C. for 12 hrs. Collected was 1.5 ml of the resulting culture and cells were harvested by centrifugation followed by washing with a 50 mM boric acid buffer solution (pH 9.0).
- the cells were suspended in 1.5 ml of the buffer solution to prepare a cell suspension.
- This cell suspension was sonicated to destruct the cells and obtain crude enzyme extract.
- the resulting crude extract was subjected to heat treatment for 30 min. at 40, 45, 50, 55, 60, and 65° C., and then immediately cooled to 4° C.
- the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity was measured according to the method described in Example 2. For each mutant, the relative remaining activity to the control (100%), which was kept cooled at 4° C. without heat treatment, was determined.
- the vertical axis represents a remaining ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity
- the horizontal axis represents a treatment temperature.
- the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase completely lost its enzyme activity at 50° C.
- the single mutants, I-2, I-23, J-05, and K-01 retained the relative remaining activities of 75% at 50° C., 85% at 50° C., 12% at 60° C., and 12% at 55° C., respectively, and it was confirmed that the heat resistance of these single mutants improved compared with that of the wild type.
- the mutation sites of the single mutants, I-2, I-23, J-05, and K-01 were the isoleucine residue at 166, the lysine residue at 120, the isoleucine residue at 166, and the alanine residue at 366, respectively.
- restriction enzymes EcoT22I, AccI, and ClaI provided were DNA fragments, each of which contains one of these mutation sites ( FIG. 6 ).
- a chimeric enzyme gene having double mutation or triple mutation was formed ( FIG. 6 ).
- Ch-4 first, pEDTrcI-23 that contained a gene encoding the single mutant I-23 was cleaved by restriction enzymes NcoI and AccI, and the shorter fragment (about 0.5 kb) of the resulting DNA fragments was extracted by agarose gel electrophoresis.
- pEDTrcJ-05 that contained a gene encoding the single mutant J-05 was cleaved by restriction enzymes NcoI and AccI, and the longer fragment (about 3.8 kb including the vector) of the resulting DNA fragments was extracted by agarose gel electrophoresis. Both of the DNA fragments were recovered by using a DNA PREP (from Diatron) and ligated by using a DNA Ligation Kit Ver. 1 (from Takara Shuzo), and then E. coli JM109 was transformed therewith according to the conventional method.
- plasmid DNA and the chimeric enzyme were named pEDTrcCh-4 and Ch-4, respectively.
- NcoI-AccI fragment about 0.5 kb
- NcoI-AccI fragment about 3.8 kb including the vector
- an NcoI-ClaI fragment (about 1 kb) from the single mutant J-05 and an NcoI-ClaI fragment (about 3.3 kb including the vector) from the single mutant K-01 were ligated to prepare plasmid DNA pEDTrcCh-1 and a chimeric enzyme Ch-1.
- An NcoI-ClaI fragment (about 1 kb) from the single mutant I-2 and an NcoI-ClaI fragment (about 3.3 kb including the vector) from the single mutant K-01 were ligated to prepare plasmid DNA pEDTrcCh-2 and a chimeric enzyme Ch-2.
- an NcoI-AccI fragment (about 0.5 kb) from the single mutant 1-23 and an AccI-ClaI fragment (about 0.5 kb) from the single mutant J-05, and an NcoI-ClaI fragment (about 3.3 kb including the vector) from the single mutant K-01 are ligated to prepare plasmid DNA pEDTrcCh-8 and a chimeric enzyme Ch-8.
- NcoI-AccI fragment (about 0.5 kb) from the single mutant I-23 and an AccI-ClaI fragment (about 0.5 kb) from the single mutant I-2, and an NcoI-ClaI fragment (about 3.3 kb including the vector) from the single mutant K-01 are ligated to prepare plasmid DNA pEDTrcCh-10 and a chimeric enzyme Ch-10.
- the structures of the resulting chimeric enzymes are shown in FIG. 6 .
- coli bearing the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase JM109/pEDTrc9003 were inoculated into 1.5 ml of an LBAmp medium to incubate with shaking at 30° C. for 8 hrs. Then, 400 ⁇ l of each of the culture solutions was inoculated into 40 ml of an LBAmp medium prepared in a 500 ml Erlenmeyer's flask to culture with shaking at 37° C. for 12 hrs. Collected was 1.5 ml of the resulting culture and cells were harvested by centrifugation followed by washing with a 50 mM boric acid buffer solution (pH 9.0).
- the cells were suspended in 1.5 ml of the buffer solution to prepare a cell suspension.
- This cell suspension was sonicated to destruct the cells and obtain a crude enzyme extract.
- the resulting crude extract was subjected to heat treatment for 30 min. at 40, 45, 50, 55, 60, and 65° C., and then immediately cooled to 4° C.
- the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity was measured according to the method described in Example 2.
- the relative remaining activity to the control which was kept cooled at 4° C. without heat treatment, was determined. The results are shown in FIG. 7 .
- FIG. 7 In FIG.
- the vertical axis represents a remaining ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity
- the horizontal axis represents a treatment temperature.
- the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase completely lost its enzyme activity at 50° C.
- the multiple mutants, Ch-4, Ch-6, Ch-1, Ch-2, Ch-8, and Ch-10 retained the relative remaining activities of 72% at 60° C., 89% at 55° C., 56% at 60° C., 25% at 55° C., 90% at 60° C., and 97% at 55° C., respectively, and it was confirmed that the heat resistance of these multiple mutants improved compared with that of the wild type and the single mutants.
- the present invention provides a nucleotide sequence and an amino acid sequence of a wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase derived from Brevundimonas diminuta strain MR-E001. Further, the present invention provides a nucleotide sequence and an amino acid sequence of a modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase derived from the wild type ethylenediamine-N,N′-disuccinate:ethylenediamine lyase.
- the present invention provides recombinant DNAs containing the wild type and the modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase genes; transformants or transductants containing the recombinant DNAs; and a method of preparing diaminoalkylene-N,N′-disuccinates by using the transformants or transductants.
- diaminoalkylene-N,N′-disuccinates can be prepared efficiently.
- SEQ ID NO: 1: Xaa represents Met or deletion
- SEQ ID NO: 3 Artificial DNA
- SEQ ID NO: 4 Artificial DNA
- SEQ ID NO: 5 Artificial DNA
- SEQ ID NO: 6 Artificial DNA
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
(4) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(5) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least the isoleucine residue at 166 with serine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(6) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least an isoleucine residue at 166 with threonine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(7) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(8) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid and an isoleucine residue at 166 with serine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(9) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid and an isoleucine residue at 166 with threonine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(10) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least an isoleucine residue at 166 with serine and an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(11) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least an isoleucine residue at 166 with threonine and an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(12) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid and an isoleucine residue at 166 with serine an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(13) A protein comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of at least a lysine residue at 120 with glutamic acid and an isoleucine residue at 166 with threonine and an alanine residue at 365 with valine, and having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(14) A gene encoding the protein according to (1).
(15) A gene of the following (a) or (b):
(a) a gene comprising a nucleotide sequence described in SEQ ID NO: 2; or
(b) a gene that hybridizes under stringent conditions to a DNA comprising a sequence complementary to a DNA comprising the nucleotide sequence of SEQ ID NO: 2 or a portion thereof, and encodes a protein having an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase activity.
(16) A gene encoding the protein according to (2).
(17) The gene according to (16) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by deletion, substitution or addition of one or more bases.
(18) A gene encoding the protein according to (3).
(19) The gene according to (18) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of at least one base of bases from 358 to 360, from 496 to 498, and from 1093 to 1095 with a different base.
(20) A gene encoding the protein according to (4).
(21) The gene according to (20) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG.
(22) The gene according to (20) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, adenine, at 358 with guanine.
(23) A gene encoding the protein according to (5).
(24) The gene according to (23) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases ATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T).
(25) The gene according to (23) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of the base, thymine, at 497 with guanine.
(26) A gene encoding the protein according to (6).
(27) The gene according to (26) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases ATC from 496 to 498 with ACN (N refers to A, G, C or T).
(28) The gene according to (26) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, thymine, at 497 with cytosine.
(29) A gene encoding the protein according to (7).
(30) The gene according to (29) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T).
(31) The gene according to (29) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, cytosine, at 1094 with thymine.
(32) A gene encoding the protein according to (8).
(33) The gene according to (32) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG, and bases ATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T).
(34) The gene according to (32) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, adenine, at 358 with guanine, and a base, thymine, at 497 with guanine.
(35) A gene encoding the protein according to (9).
(36) The gene according to (35) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG, and bases ATC from 496 to 498 with ACN (N refers to A, G, C or T), respectively.
(37) The gene according to (35) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, adenine, at 358 with guanine, and a base, thymine, at 497 with cytosine, respectively.
(38) A gene encoding the protein according to (10).
(39) The gene according to (38) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases ATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T), and bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T), respectively.
(40) The gene according to (38) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, thymine, at 497 with guanine, and a base, cytosine, at 1094 with thymine, respectively.
(41) A gene encoding the protein according to (11).
(42) The gene according to (41) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases ATC from 496 to 498 with ACN (N refers to A, G, C or T), and bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T), respectively.
(43) The gene according to (41) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, thymine, at 497 with cytosine, and a base, cytosine, at 1094 with thymine, respectively.
(44) A gene encoding the protein according to (12).
(45) The gene according to (44) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG, bases ATC from 496 to 498 with AGC, AGT, or TCN (N refers to A, G, C or T), and bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T), respectively.
(46) The gene according to (44) comprising a nucleotide sequence wherein the base, adenine, at 358 is substituted with guanine, the base, thymine, at 497 with guanine, and the base, cytosine, at 1094 with thymine in the nucleotide sequence of SEQ ID NO: 2.
(47) A gene encoding the protein according to (13).
(48) The gene according to (47) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of bases AAA from 358 to 360 with GAA or GAG, bases ATC from 496 to 498 with ACN (N refers to A, G, C or T), and bases GCC from 1093 to 1095 with GTN (N refers to A, G, C or T), respectively.
49) The gene according to (47) comprising a nucleotide sequence derived from the nucleotide sequence of SEQ ID NO: 2 by substitution of a base, adenine, at 358 with guanine, a base, thymine, at 497 with cytosine, and a base, cytosine, at 1094 with thymine, respectively.
(50) A recombinant wherein the gene DNA according to any one of (14) to (49) is inserted into a DNA vector.
(51) A transformant or transductant comprising the recombinant DNA according to (50).
(52) A method of producing an ethylenediamine-N,N′-disuccinate:ethylenediamine lyase, comprising a step of culturing the transformant or transductant according to (51) to collect the ethylenediamine-N,N′-disuccinate:ethylenediamine lyase from the resulting culture.
(53) A method of producing a diaminoalkylene-N,N′-disuccinate, comprising a step of reacting fumaric acid and a diamine in the presence of the transformant or transductant according to (51) to collect the diaminoalkylene-N,N′-disuccinate from the resulting reaction products.
TABLE 1 |
Bacteriological properties of MR-E001 strain |
Morphology | rod-shaped | ||
Gram stain | − | ||
Spores | − | ||
Mobility | + | ||
Flagella | polar flagellation | ||
Oxygen requirement | aerobic | ||
Oxidase | + | ||
Catalase | + | ||
OF test | − | ||
Color tone of colonies | No characteristic pigment | ||
is generated. | |||
Production of fluorescent pigment | − | ||
Accumulation of PHB | + | ||
Auxotrophy | present | ||
Quinone system | Q-10 | ||
Reduction of nitrates | + | ||
Production of Indole | − | ||
Arginine dihydrolase | − | ||
Urea degradation | − | ||
Esculin degradation | − | ||
Gelatin liquefaction | − | ||
PNPG | − | ||
Assimilation | |||
Glucose | − | ||
L-Arabinose | − | ||
D-Mannose | − | ||
D-Mannitol | − | ||
N-Acetyl-D-glucosamine | − | ||
Maltose | − | ||
Potassium gluconate | + | ||
n-Capric acid | − | ||
Adipic acid | + | ||
dl-Malic acid | + | ||
Citric acid | + | ||
Phenyl acetate | − | ||
(SEQ ID NO: 3) |
Primer #1: ATGACICCIC AYAAYCCIGA YGC | |
(SEQ ID NO: 4) |
Primer #2: CCDATYTGCAT YTTICCIGC RACIGAICCD ATYTC |
(SEQ ID NO: 5) |
ED-01: CGCCATGGCC CCGCATAACC CAGATGCCAC C |
(The underlined part is a cleavage recognition site by the restriction enzyme NcoI); and
(SEQ ID NO: 6) |
ED-02: AAACAAGCTT CGTCATGGCT ATCCCCTC |
(The underlined part is a cleavage recognition site by the restriction enzyme HindIII). Composition of reaction solution:
Template DNA (pEDS9020 prepared in the above step) | 1 | |
10× PCR buffer (from GIBCO) | 10 | |
50 mM MgCl2 (from GIBCO) | 3 | μl |
Primer ED-01 | 1 | μl |
Primer ED-02 | 1 | μl |
2.5 |
2 | μl each |
10 |
2 | |
10 |
2 | μl |
Sterile water | 71 | μl |
Taq DNA polymerase (from GIBCO) | 1 | μl. |
Template DNA (pEDS9020) | 1 | |
10× Pyrobest Buffer (from Takara Shuzo) | 10 | μl |
Primer ED-01 | 1 | μl |
Primer ED-02 | 1 | μl |
5 |
2 | μl each |
Sterile water | 78 | μl |
Pyrobest ™ DNA polymerase (from Takara Shuzo) | 1 | μl. |
Claims (12)
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/691,443 US8728792B2 (en) | 2004-05-20 | 2010-01-21 | Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase |
US13/547,973 US8895285B2 (en) | 2004-05-20 | 2012-07-12 | Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase |
US14/243,743 US9212356B2 (en) | 2004-05-20 | 2014-04-02 | Modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/JP2004/007226 WO2005113766A1 (en) | 2004-05-20 | 2004-05-20 | Modified ethylenediamine-n,n’-disuccinic acid:ethylenediamine lyase |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2004/007226 A-371-Of-International WO2005113766A1 (en) | 2004-05-20 | 2004-05-20 | Modified ethylenediamine-n,n’-disuccinic acid:ethylenediamine lyase |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/691,443 Division US8728792B2 (en) | 2004-05-20 | 2010-01-21 | Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase |
Publications (2)
Publication Number | Publication Date |
---|---|
US20080009053A1 US20080009053A1 (en) | 2008-01-10 |
US7674611B2 true US7674611B2 (en) | 2010-03-09 |
Family
ID=35428405
Family Applications (4)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US11/569,339 Expired - Fee Related US7674611B2 (en) | 2004-05-20 | 2004-05-20 | Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase |
US12/691,443 Expired - Fee Related US8728792B2 (en) | 2004-05-20 | 2010-01-21 | Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase |
US13/547,973 Expired - Fee Related US8895285B2 (en) | 2004-05-20 | 2012-07-12 | Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase |
US14/243,743 Expired - Fee Related US9212356B2 (en) | 2004-05-20 | 2014-04-02 | Modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/691,443 Expired - Fee Related US8728792B2 (en) | 2004-05-20 | 2010-01-21 | Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase |
US13/547,973 Expired - Fee Related US8895285B2 (en) | 2004-05-20 | 2012-07-12 | Modified ethylenediamine-N, N′-disuccinate: ethylenediamine lyase |
US14/243,743 Expired - Fee Related US9212356B2 (en) | 2004-05-20 | 2014-04-02 | Modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase |
Country Status (4)
Country | Link |
---|---|
US (4) | US7674611B2 (en) |
EP (1) | EP1757685B1 (en) |
DE (1) | DE602004019824D1 (en) |
WO (1) | WO2005113766A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005113766A1 (en) | 2004-05-20 | 2005-12-01 | Mitsubishi Rayon Co., Ltd. | Modified ethylenediamine-n,n’-disuccinic acid:ethylenediamine lyase |
JP5504514B2 (en) * | 2009-02-11 | 2014-05-28 | 国立大学法人島根大学 | Method for producing L-aspartic acid |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0845536A2 (en) | 1996-11-29 | 1998-06-03 | Nitto Chemical Industry Co., Ltd. | Protein having ethylenediamine-N,N'-disuccinic acid: ethylenediamine lyase activity and gene encoding the same |
EP0927762A2 (en) | 1997-10-28 | 1999-07-07 | Mitsubishi Rayon Co., Ltd. | Method for removing fumarase activity, Microorganisms obtainable by the method and production of optically active aminopolycarboxylic acids using the microorganisms |
EP1043400A1 (en) | 1997-12-22 | 2000-10-11 | Mitsubishi Rayon Co., Ltd. | Process for producing [s,s]-ethylenediamine-n,n'-disuccinic acid |
EP1174515A1 (en) | 1999-04-27 | 2002-01-23 | Mitsubishi Rayon Co., Ltd. | Processes for producing s,s-2-hydroxypropylenediamine-n,n'-disuccinic acid |
WO2002006442A2 (en) | 2000-07-19 | 2002-01-24 | Novozymes A/S | Cell-wall degrading enzyme variants |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005113766A1 (en) * | 2004-05-20 | 2005-12-01 | Mitsubishi Rayon Co., Ltd. | Modified ethylenediamine-n,n’-disuccinic acid:ethylenediamine lyase |
-
2004
- 2004-05-20 WO PCT/JP2004/007226 patent/WO2005113766A1/en active Application Filing
- 2004-05-20 DE DE602004019824T patent/DE602004019824D1/en not_active Expired - Lifetime
- 2004-05-20 US US11/569,339 patent/US7674611B2/en not_active Expired - Fee Related
- 2004-05-20 EP EP04734134A patent/EP1757685B1/en not_active Expired - Fee Related
-
2010
- 2010-01-21 US US12/691,443 patent/US8728792B2/en not_active Expired - Fee Related
-
2012
- 2012-07-12 US US13/547,973 patent/US8895285B2/en not_active Expired - Fee Related
-
2014
- 2014-04-02 US US14/243,743 patent/US9212356B2/en not_active Expired - Fee Related
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6168940B1 (en) | 1996-11-12 | 2001-01-02 | Nitto Chemical Industry Co., Ltd. | Protein having ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase acitivity and gene encoding the same |
EP0845536A2 (en) | 1996-11-29 | 1998-06-03 | Nitto Chemical Industry Co., Ltd. | Protein having ethylenediamine-N,N'-disuccinic acid: ethylenediamine lyase activity and gene encoding the same |
EP0927762A2 (en) | 1997-10-28 | 1999-07-07 | Mitsubishi Rayon Co., Ltd. | Method for removing fumarase activity, Microorganisms obtainable by the method and production of optically active aminopolycarboxylic acids using the microorganisms |
EP1043400A1 (en) | 1997-12-22 | 2000-10-11 | Mitsubishi Rayon Co., Ltd. | Process for producing [s,s]-ethylenediamine-n,n'-disuccinic acid |
EP1174515A1 (en) | 1999-04-27 | 2002-01-23 | Mitsubishi Rayon Co., Ltd. | Processes for producing s,s-2-hydroxypropylenediamine-n,n'-disuccinic acid |
US6503739B1 (en) | 1999-04-27 | 2003-01-07 | Mitsubishi Rayon Co., Ltd. | Processes for producing S,S-2-hydroxypropylenediamine-N-N′-disuccinic acid |
WO2002006442A2 (en) | 2000-07-19 | 2002-01-24 | Novozymes A/S | Cell-wall degrading enzyme variants |
Non-Patent Citations (7)
Title |
---|
"Directed evolution of a thermostable esterase" by Giver et al., Proc. Natl. Acad. Sci. USA, vol. 95, pp. 12809-12813, Oct. 1998. |
"Random-priming in vitro recombination: an effective tool for directed evolution" by Shao et al., Nucleic Acids Research, 1998, vol. 26, No. 2, pp. 681-683. |
Branden et al. Introduction to protein structure, Gerald Publishing Inc., New York, p. 247, 1991. * |
International Search Report for PCT/JP2004/007226 mailed Jul. 6, 2004. |
Seffernick et al. Melamine deaminase and atrazine chlorohydrolase: 98 percent identical but functionally different, J Bacteriol. Apr. 2001;183(8):2405-10. * |
Segers et al., Intl. J. Syst. Bacteriol, 1994, vol. 44, No. 3, p. 499-510. |
Witkowski et al. Conversion of a beta-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine, Biochemistry. Sep. 7, 1999;38(36):11643-50. * |
Also Published As
Publication number | Publication date |
---|---|
US20130184448A1 (en) | 2013-07-18 |
US9212356B2 (en) | 2015-12-15 |
US8895285B2 (en) | 2014-11-25 |
DE602004019824D1 (en) | 2009-04-16 |
EP1757685A4 (en) | 2007-08-01 |
US20080009053A1 (en) | 2008-01-10 |
WO2005113766A1 (en) | 2005-12-01 |
US20100151537A1 (en) | 2010-06-17 |
US8728792B2 (en) | 2014-05-20 |
US20140248672A1 (en) | 2014-09-04 |
EP1757685B1 (en) | 2009-03-04 |
EP1757685A1 (en) | 2007-02-28 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US7645605B2 (en) | Heat-resistant nitrile hydratase | |
JP3692538B2 (en) | Novel lysine decarboxylase gene and method for producing L-lysine | |
KR100723826B1 (en) | Mutant -aminotransferase and process for producing optically active glutamic acid derivative using the same | |
EP0955368B1 (en) | L-glutamic acid-producing bacterium and method for producing l-glutamic acid | |
US7098019B2 (en) | DNA for encoding D-hydantoin hydrolases, DNA for encoding N-carbamyl-D-amino acid hydrolases, recombinant DNA containing the genes, cells transformed with the recombinant DNA, methods for producing proteins utilizing the transformed cells and methods for producing D-amino acids | |
JPH11155571A (en) | Production of l-cysteine | |
US8460902B1 (en) | DNA encoding hydantoinase, DNA encoding N-carbamyl-L-amino acid hydrolase, recombinant DNA, transformed cell, method of producing protein, and method of producing optically active amino acid | |
JP2001046067A (en) | L-lysine biosynthetic gene derived from thermophilic bacillus bacterium | |
US7118898B1 (en) | Rhodococcus bacterium, nitrilase gene, nitrylhydratase gene and amidase gene from Rhondococcus bacterium, and process for producing carboxylic acids by using them | |
US9212356B2 (en) | Modified ethylenediamine-N,N′-disuccinate:ethylenediamine lyase | |
EP1616945B1 (en) | Process for producing D-N-carbamoyl-alpha-amino acids | |
US20030148472A1 (en) | DNA for encoding D-hydantoin hydrolases, DNA for encoding N-carbamyl-D-amino acid hydrolases, recombinant DNA containing the genes, cells transformed with the recombinant DNA, methods for producing proteins utilizing the transformed cells and methods for producing D-amino acids | |
NZ250369A (en) | Dna from bacteria which confers on a riboflavin requiring host microorganism the ability to complement its riboflavin requirement; recombinant dna and microorganisms containing such dna | |
JP2004215513A (en) | Improved nitrile hydratase | |
JP4253195B2 (en) | Modified ethylenediamine-N, N'-disuccinic acid: ethylenediamine lyase | |
EP1002866A1 (en) | TEMPERATURE SENSITIVE dtsR GENES | |
KR20020087948A (en) | DNA Encoding Novel D-Aminoacylase and Process for Producing D-Amino Acid by Using the Same | |
US6168940B1 (en) | Protein having ethylenediamine-N,N′-disuccinic acid:ethylenediamine lyase acitivity and gene encoding the same | |
KR100869687B1 (en) | Process for producing l-amino acid via fermentation method | |
JP2004222538A (en) | Advanced nitrile hydratase | |
EP1712627B1 (en) | Polypeptide having amidase activity and gene thereof | |
JP4997420B2 (en) | Mutant microorganism |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: MITSUBISHI RAYON CO., LTD., JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:AKIYAMA, TAKANORI;MIZUNASHI, WATARU;NAKAMURA, TETSUJI;REEL/FRAME:018739/0405 Effective date: 20061127 Owner name: MITSUBISHI RAYON CO., LTD.,JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:AKIYAMA, TAKANORI;MIZUNASHI, WATARU;NAKAMURA, TETSUJI;REEL/FRAME:018739/0405 Effective date: 20061127 |
|
FEPP | Fee payment procedure |
Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
MAFP | Maintenance fee payment |
Free format text: PAYMENT OF MAINTENANCE FEE, 8TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1552) Year of fee payment: 8 |
|
AS | Assignment |
Owner name: MITSUBISHI CHEMICAL CORPORATION, JAPAN Free format text: CHANGE OF NAME;ASSIGNOR:MITSUBISHI RAYON CO., LTD.;REEL/FRAME:043750/0834 Effective date: 20170401 |
|
FEPP | Fee payment procedure |
Free format text: MAINTENANCE FEE REMINDER MAILED (ORIGINAL EVENT CODE: REM.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
LAPS | Lapse for failure to pay maintenance fees |
Free format text: PATENT EXPIRED FOR FAILURE TO PAY MAINTENANCE FEES (ORIGINAL EVENT CODE: EXP.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY |
|
STCH | Information on status: patent discontinuation |
Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362 |
|
FP | Lapsed due to failure to pay maintenance fee |
Effective date: 20220309 |